Use of acylsulfonamido-substituted polymethine dyes as fluorescene dyes and/or markers

ABSTRACT

The invention relates to a novel polymethine dyes containing at least one acylsulfonamido group of the formula (I) 
                 
 
in which n, Y, A and R have the meaning given in the claims, and at least one compound of the formulae (a) to (n) given in the claims. The polymethine dyes according to the invention are suitable for use as dyes and/or labels, in particular for staining or labeling biomolecules.

In recent years, more and more sensitive and easy to handle detection methods have been developed which are used, in particular, in biotechnology where only small amounts of substance are available for an analysis. Here, for example, fluorescent detection methods are finding increasingly widespread use. The fluorescent dyes are used as labels to label biological substances such as proteins, DNA, RNA, carbohydrates, fats or entire cells. However, because many of the substances to be labeled for their part have fluorescent properties in the range from 400 to 500 nm, the dyes used for labeling should absorb at wavelength above 500 nm, usually in a range between 500 and 1,200 nm. Moreover, in this wavelength region, inexpensive laser diols are available as light sources.

The fluorescent dyes known from the prior art are frequently substituted by hydrophilic or acidic groups to ensure solubility in water at neutral or slightly basic pH. This facilitates the labeling of biological material which is operational mainly in aqueous medium. Moreover, the dyes should be photostable to allow even measurement methods which comprise relatively long irradiation. However, many of the dyes which absorb at a relatively long wavelength do not have sufficient photostability.

The dyes are bound covalently or else by absorption to the biological samples. The sulfhydryl groups and free amino groups of proteins, for example, are reactive radicals which allow covalent coupling a dye molecule. Furthermore, hydrophilic or lipophilic dyes can add themselves by absorption to the hydrophilic and hydrophobic domains of the proteins. It is also possible for ionic dyes to be absorbed to proteins by ionic interactions.

The cyanine dyes (Waggoner et al. Bioconjugate Chemistry, 4, 105-111, (1993), U.S. Pat. No. 5,268,486, WO 97/13810) which have hitherto frequently been used in bioassays tend to form aggregates. In addition to the monomer, H aggregates and J aggregates may from, depending on the chemical environment (for example pH) of the dye. This is exploited in photography, since the absorptions of the aggregates are shifted hypsochromically or bathochromically compared to the absorption of the monomer. However, for biolabeling, this aggregation effect is undesirable since here, too, one or more additional absorption bands result.

Frequently, multiplex assays are carried out where samples labeled with different dyes are excited at different wavelengths. To achieve a satisfactory separation of excitation and emission signals, the absorptions of the dye conjugates must not overlap. Here, a sharp, reproducible and pH- and solvent-independent absorption of the dyes is important. The tendency of the frequency used cyanine dyes to form aggregates furthermore reduces the intensity of the fluorescence by quenching. The emitted radiation is either directly reabsorbed, or there are radiationless energy losses.

The dyes known from the prior art for biolabeling, i.e. the cyanine dyes, are a class of the polymethines. In addition, WO 97/40104 discloses, for example, squaric acid derivatives as dyes for biolabeling.

It was an object of the present invention to provide novel fluorescent dyes as fluorescent labels or dyes for staining having improved properties compared to the cyanine dyes known from the prior art. In particular, solubility in biological buffer systems and photostability were to be improved and, in particular, the tendency to form aggregates to be reduced. However, at the same time, the compounds according to the invention were also to have a high quantum yield and a higher extinction coefficient.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the photostability of three dyes tested in the Examples.

The present invention provides polymethine dyes containing at least one acylsulfonamido group according to formula (I)

in which

-   n represents 1, 2, 3, 4, 5, 6, 7 or 9 -   Y, A represent electron-withdrawing radicals, preferably C═O, —SO2—,     and -   R represents an optionally substituted alkyl or aryl radical     and containing at least one compound of the formulae below:

The present invention furthermore provides the use of polymethine dyes containing at least one acrylsulfonamido group according to formula (I) for staining and/or biolabeling of biomolecules.

Polymethine dyes according to the invention which are to be used not only for staining but also for biolabeling by forming a covalent bond have, in addition to a group of the formula (I), additionally at least one group capable of forming such a covalent bond; according to the invention, such groups are preferably

Accordingly, the present invention furthermore provides the use of polymethine dyes containing at least one acylsulfonamido group according to formula (I) and at least one grouping capable of forming a covalent bond selected from the list of the following compounds:

for labeling biomolecules by covalent binding.

For the purpose of the present application, biomolecules are to be understood as meaning proteins and also DNA and/or RNA. Moreover, for the purpose of the present application, cells, too, may be considered as biomolecules. Also included are small organic molecules having biological action.

Polymethine dyes, which, in addition to a compound of the formula (I), additionally have a group selected from one of the formulae (a) to (n) are hitherto not known in the prior art and represent novel compounds. They are suitable, in particular, for biolabeling, as they are capable of forming covalent bonds.

For the purpose of the present application, an electron-withdrawing group is preferably to be understood as meaning, unless described otherwise, groups as described in March, Advanced Organic Chemistry, 3rd Ed., p. 17 and p. 238.

For the purpose of the present application, alkyl is, unless defined otherwise, to be understood as meaning linear or branched, cyclic or straight-chain, substituted or unsubstituted hydrocarbons. These are in particular alkyl groups having 1 to 12 C atoms, such as, for example, methyl ethyl, propyl, isopropyl, butyl, t-butyl, neopentyl and 2-ethylhexyl groups. However, these can be substituted further, particularly preferably by a carboxycarbonyl group.

For the purpose of the present application, aryl is, unless defined otherwise, to be understood as meaning aromatic hydrocarbon groups, preferably 5- or 6-membered ring systems, which may be present in monocyclic from or else as condensed ring systems. These can be either substituted or else unsubstituted ring systems. Particular preference is given, for example, to phenyl and naphthyl groups.

Surprisingly, it has been found that, when the dyes according to the invention are used in biological systems, such as, for example, in protein conjugates or in biological buffer media, the tendency of the polymethines to aggregates could be reduced considerably compared to cyanines or cyanines or squaric acid dyes with alkylsulfonates or alkylcarboxylates, as described in the patents U.S. Pat. No. 5,268,486, WO 96/00902, WO 97/13810 and WO 97/40104.

A further advantage of the dyes to be used according to the invention is the fact that the chromogenic properties of the polymethines are not changed by the substituent. Hitherto, for example, the functionality for an improved solubility of the cyanine dyes has been realized by introducing sulfo groups into the aryl system which is part of the chromophore. This also resulted in a shift of the spectral properties, since the substituents had electron-withdrawing effects. The spectral properties of unsubstituted arylcyanines have been described and it is thus possible, by introducing the acylsulfonic acid radical, to adapt the dye system directly for use in a bioassay, for example. Thus, time-consuming optimization and screening experiments can be dispensed with.

Furthermore, compared to alkylcarboxylates, the dyes having acylsulfonamido groups have a high extinction coefficient. This results in a further advantage when the dye is used as a label:

If the dyes are bound covalently as a label, for example to a protein or an antibody, the conjugate is characterized by the molar dye/protein content ratio. In order to obtain a satisfactory signal, a certain amount of dye has to be bound to the protein. The higher the extinction coefficient of the dye and thus its intensity, the higher the sensitivity of the dye as a fluorescent label, since the intensity of the fluorescence at absorptions <0.05 is a function of the extinction coefficient.

-   -   F=φKIoεcd     -   φ=quantum yield     -   Io=intensity of the light with which the sample is irradiated     -   ε=extinction coefficient     -   c=concentration of the sample     -   d=path length

However, since dyes quench when they are spatially close, the dye/protein ratio should not be too high. This would result in a loss of fluorescence intensity. Accordingly, dyes having high extinction coefficients are particularly suitable for use as fluorescent labels. Even small amounts of dye give an intensive fluorescence signal at a low molar dye/protein content ratio. Also, quench processes are minimized.

Suitable polymethine dyes to be used according to the invention are, in particular, cyanine dyes, merocyanines, rhodacyanines, styrene dyes, squaric acid dyes and the crotonic acid dyes, as described hereinbelow in more detail in preferred embodiments, which are all characterized in that, for the use according to the invention, they have at least one substituent according to the formula (I). In a further embodiment, the polymethine dyes of the formula (I) to (5) may also be attached to one another, it being possible for both two or more identical or else different dyes to be attached to one another. Preferred polymethine dyes according to the invention are those of the formulae shown below which, in addition to a substituent of the formula (I), contain additionally at lest one substituent selected from the group of the compounds below:

1. Cyanine Dyes

According to the invention, suitable cyanine dyes are in particular those of the formulae 1a, 1b and/or 1c

in which

-   Ra₁₀ to Ra₁₁₀ independently of one another may represent any     substituent, -   n represents 0, 1, 2, 3 or 4, -   Ya₁₁, Ya₁₂ and Ya₁₃ independently of one another may represent     substituted or unsubstituted C or N, where the substituents may also     form a 5- or 6-membered aliphatic or aromatic carbo- or heterocycle,     and -   Aa₁₁ and Ba₁₁ independently of one another represent O, S, Se, T,     N-Ra₁₁₁, C(Ra₁₁₂)(Ra₁₁₃) or —C(Ra₁₁₄)═C(Ra₁₁₅)—, where Ra₁₁ to Ra₁₁₅     independently of one another represent H, optionally substituted     alkyl, optionally substituted aryl or optionally substituted alkenyl     having up to 20 C atoms, with the proviso that at least one of the     substituents Ra10 to Ra115 is selected from the group consisting of     —(CH₂)₁—SO₂—Z—SO₂—R, —(CH₂)₁—CO—Z—SO₂—R, —(CH₂)₁—SO₂—Z—CO—R and     —(CH₂)₁—N(R)—SO₃ ⁻,     where -   1 is a number between 1 and 6, -   Z represents NH or N⁻ and -   R preferably represents optionally substituted alkyl or aryl,     Particularly preferred compounds of formula 1a to be used according     to the invention are listed below     Preferred compounds of the formula 1a     in which -   Rb₁₀ to Rb₁₁₀ independently of one another may represent any     substituent, -   m represents 0, 1, 2, 3, or 4 and -   Yb₁₁, Yb₁₂ and Yb₁₃ independently of one another may represent     substituted or unsubstituted C or N, where the substituents may also     form a 5- or 6-membered aliphatic or aromatic carbo- or heterocycle,     with the proviso that at least one of the substituents Rb₁₁ to Rb₁₁₀     is selected from the group consisting of —(CH₂)_(i)—SO₂—Z—SO₂—R,     —(CH₂)₁—CO—Z—SO₂—, —(CH₂)₁—SO₂—Z—CO—R and —(CH₂)₁—N(R)—SO₃ ⁻,     where -   1 represents a number between 1 and 6, -   Z represents NH or N⁻ and -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the following substituents form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

The substituent Rb₁₁ together with Rb₁₂ and/or Rb₁₄, the substituent Rb₁₂ together with Rb₁₃ and/or Rb₁₁, the substituent Rb₁₄ together with Rb₁₅ and/or Rb₁₁, the substituent Rb₁₈ together with Rb₁₉ and/or Rb₁₇, the substituent Rb₁₇ together with Rb₁₆ and/or R₁₈, the substituent Rb₁₉ together with R₁₁₀ and/or Rb₁₈. Particularly preferred compounds of the formula 1b

in which

-   Rc₁₁ to Rc₁₁₀ independently of one another may represent any     substituent, -   m represents 0, 1, 2, 3 or 4, -   Yc₁₁, Yc₁₂, and Yc₁₃ independently of one another may represent     substituted or unsubstituted C or N, where the substituents may also     form a 5- or 6-membered aliphatic or aromatic carbo- or heterocycle.     and -   Bc₁₁ represents O, S, Se, Te, N-Rc₁₁₁, C(Rc₁₁₂)(Rc₁₁₃) or     —C(Rc₁₁₄)═C(Rc₁₁₅)—, where Rc₁₁ to Rc₁₁₅ independently of one     another may represent H, optionally substituted alkyl, optionally     substituted aryl or optionally substituted alkenyl having up to 20 C     atoms, with the proviso that at least one of the substituents Rc10     to Rc115 is selected from the groups consisting of     —(CH₂)₁—SO₂—Z—SO₂—R, —(CH₂)₁—CO—Z—SO₂—R, —(CH₂)₁—SO₂—Z—CO—R and     —(CH₂)₁—N(R)—SO₃ ⁻,     where -   1 represents a number 1 and 6, -   Z represents NH or N⁻ and -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the following substituents form a 5- or 6-membered carbo- or heterocycle which is optionally also fused;

The substituent Rc₁₁ together with Rc₁₂ and/or Rc₁₄, the substituent Rc₁₂ together with Rc₁₃ and/or Rc₁₁, the substituent Rc₁₄ together with Rc₁₅ and/or Rc₁₁.

The substituents Rc₁₇ to Rc₁₁₀ may preferably form the remaining members of a carbo- or heterocyclic ring system which may contain up to 4 rings which may optionally carry a plurality of substituents. Rc₁₈ together with Rc₁₉ may form a π bond and Rc₁₇ and Rc₁₁₀ may represent substituents. Particularly preferred compounds of the formula 1c

2. Merocyanines

Particularly suitable for the purpose of the present invention are merocyanines according to formulae 2a and/or 2b

in which

-   Ra₂₁ and Ra₂₅ independently of one another represents any     substituent, -   Ya₂₁, Ya₂₂, Ya₂₃, Ya₂₄ independently of one another represent     substituted or unsubstituted C or N, where the substituents may also     form a 5- or 6-membered aliphatic or aromatic carbo- or heterocycle. -   m represents 0, 1, 2, 3, 4 -   o represents 0, 1, 2 -   Fa₂₁ and Ca₂₁ independently of one another represent C═O, C═S or     C(Ra₂₇)═, -   Ba₂₁ and Da₂₁ independently of another represent optionally     substituted C, N, O or S, -   Ra₂₆ represents O, S, or a further heterocyclic, optionally     substituted, 5- or 6-membered ring -   k, i and j independently of one another represent 0 or 1 -   Ra₂₇, represents H or optionally substituted alkyl or aryl, -   Aa₂₁ represents O, S, Se, Te, N-Ra₂₈, C(Ra₂₉)(Ra₂₁₀),     —C(Ra₂₁₁)═C(Ra₂₁₂)—,     where -   Ra₂₈ to Ra₂₁₂ may represent H or optionally substituted alkyl, aryl,     or alkenyl having up to 20 C atoms,     with the proviso that at least one of the substituents Ra₂₁ to Ra₂₅     or Ra₂₇ to Ra₂₁₂ is selected from the group consisting of     —(CH₂)₁—SO₂—Z—SO₂—R, —(CH₂)₁—CO—Z—SO₂—R, —(CH₂)₁—SO₂—Z—CO—R and     —(CH₂)₁—N(R)—SO₃ ⁻,     where -   1 represents a number between 1 and 6, -   Z represents NH or N⁻ and -   R preferably represents optionally substituted alkyl or aryl.

The substituents Ra₂₂ to Ra₂₅ preferably form the remaining members of a carbo- or heterocyclic ring system which may contain up to 4 rings which may optionally carry a plurality of substituents. Ra₂₂ together with Ra₂₅ may form a π bond and Ra₂₃ and Ra₂₄ may represent substituents. Particularly preferred compounds of the formula 2a

in which

-   -   Rb₂₁ to Rb₂₅ independently of one another represent any         substituent,     -   Yb₂₁, Yb₂₂, Yb₂₃, Yb₂₄ independently of one another represent         substituted or unsubstituted C or N, where the substituents may         also form a 5- or 6-membered aliphatic or aromatic carbo- or         heterocycle.     -   m represents 0, 1, 2, 3 or 4     -   o represents 0, 1, 2, 3 or 4     -   Fb₂₁ and Cb₂₁ independently of one another C═O, C═S or C(Rb₂₇)═,     -   Bb₂₁ and Db₂₁ independently of one another represents optionally         substituted C, N, O or S,     -   Rb₂₆ represents O, S, or a further heterocyclic, optionally         substituted, 5- or 6-membered ring     -   k, i, and j independently of one another represent 0 or 1     -   Rb₂₇ represents H or optionally substituted alkyl or aryl,

The following substituents may preferably form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

The substituent Rb₂₃ together with Rb₂₄ and/or Rb₂₂, the substituent Rb₂₂ together with Rb₂₁ and/or Rb₂₃, the substituent Rb₂₄ together with Rb₂₅ and/or Rb₂₃.

-   -   Ab₂₁: O S, Se, Te, N—Rb₂₈, C(Rb₂₉)(Rb₂₁₀), —C(Rb₂₁₁)═C(Rb₂₁₂)—,     -   Rb₂₈ to Rb₂₁₂ may be hydrogen, optionally substituted alkyl or         optionally substituted aryl, optionally substituted alkenyl,         having up to 20 C atoms, or one of the substituents listed         below,     -   with the proviso that at least one of the substituents Rb₂₁ to         Rb₂₅ or Rb₂₇ to Rb₂₁₂ is selected from the group consisting of         —(CH₂)_(l)—SO₂—Z—SO₂—R, —(CH₂)_(l)—CO—Z—SO₂—R,         —(CH₂)_(l)—SO₂—Z—CO—R and —(CH₂)₁—N(R)—SO₃ ⁻,         where     -   l represents a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.         Particularly preferred compounds of the formula 2b         I-b5         Rhodacyanines

For the purpose of the present application, the rhodacyanines used are in particular those of the formula 3a and/or 3b.

in which

-   -   Ra₃₁ to Ra₃₁₁ independently of one another represent any         substituent,     -   Ya₃₁, Ya₃₂, Ya₃₃, Ya₃₄, Ya₃₅ independently of one another         represent substituted or unsubstituted C or N, where the         substituents may also form a 5- or 6-membered aliphatic or         aromatic carbo- or heterocycle,     -   p and r independently of one another represent 0, 1 or 2     -   Qa₃₁ represents O; N(Ra₃₁₂), S or Se     -   Aa₃₁, Ba₃ independently of one another represent O, S, Se, Te,         N—Ra₃₁₃, C(Ra₃₁₄)(Ra₃₁₅) or —C(Ra₃₁₆)═C(Ra₃₁₇) where Ra₃₁₂ to         Ra₃₁₇ may represent H, optionally substituted alkyl, aryl or         alkenyl, having up to 20 C atoms, or any substituent.     -   with the proviso that at least one of the substituents Ra₃₁ to         Ra₃₁₇ is selected from the group consisting of         —(CH₂)_(l)—SO₂—Z—SO₂—R, —(CH₂)_(l)—CO—Z—SO₂—R,         —(CH₂)_(l)—SO₂—Z—CO—R and —(CH₂)_(l)—N(R)—SO₃ ⁻,         where     -   l represents a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the substituents Ra₃₂ to Ra₃₅ or Ra₃₆ to Ra₃₉ form the remaining members of a carbo- or heterocyclic ring system which may contain up to 4 rings which may optionally carry a plurality of substituents. Ra₃₂ together with Ra₃₅ and/or Ra₃₆ together with Ra₃₉ may form a π bond and Ra₃₃ and Ra₃₄ or Ra₃₇ and Ra₃₈ may represent substituents. Particularly preferred compounds of the formula 3a

in which

-   -   Rb₃₁ to Rb₃₁₁ independently of one another represent any         substituent,     -   Yb₃₁, Yb₃₂, Yb₃₃, Yb₃₄, Yb₃₅ independently of one another         represent substituted or unsubstituted C or N, where the         substituents may also form a 5- or 6-membered aliphatic or         aromatic carbo- or heterocycle,     -   p and r independently of one another represent 0, 1 or 2     -   Qb₃₁ represents O; N(Rb₃₁₂), S or Se     -   Ab₃₁, Bb₃₁ independently of one another represent O, S, Se, Te,         N—Rb₃₁₃, C(Rb₃₁₄)(Rb₃₁₅) or —C(Rb₃₁₆)═C(Rb₃₁₇), where Rb₃₁₁ to         Rb₃₁₇ may represent H, optionally substituted alkyl, aryl or         alkenyl, having up to 20 C atoms, or any substituent,     -   with the proviso that at least one of the substituents Rb₃₁ to         Rb₃₁₇ is selected from the group consisting of         —(CH₂)_(l)—SO₂—Z—SO₂—R, —(CH₂)_(l)—CO—Z—SO₂—R,         —(CH₂)_(l)—SO₂—Z—CO—R and —(CH₂)_(l)—N(R)—SO₃ ⁻,         where     -   l represents a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the substituents Rb₃₆ to Rb₃₉ form the remaining members of a carbo- or heterocyclic ring system which may contain up to 4 rings which may optionally carry a plurality of substituents. Rb₃₆ together with Rb₃₉ may form a π bond and Ra₃₇ and Ra₃₈ may represent substituents. The following substituents may form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

The substituent Rb₃₃ together with Rb₃₂ and/or Rb₃₄, the substituent Rb₃₂ together with Rb₃₃ and/or Rb₃₁, the substituent Rb₃₄ together with Rb₃₅ and/or Rb₃₃. Particularly preferred compounds of the formula 3b

Styryl Dyes

Styryl dyes which are to be used particularly preferably for the purpose of the present invention are the compounds of the formula 4a, 4b, 4c and/or 4d.

in which

-   -   Ra₄₁ to Ra₄₁₁ independently of one another represent any         substituent,     -   Ya₄₁ and Ya₄₂ independently of one another represent substituted         or unsubstituted C or N, where the substituents may also form a         5- or 6-membered aliphatic or aromatic carbon- or heterocycle.     -   Aa₄ represents O, S, Se, Te, N—Ra₄₁₂, C(Ra₄₁₃)(Ra₄₁₄) or         —C(Ra₄₁₅)═C(Ra₄₁₆)—, where Ra₄₁₂ to Ra₄₁₆ may represent H,         optionally substituted alkyl, aryl, or alkenyl, having up to 20         carbon atoms, or any substituent,     -   with the proviso that at least one of the substituents Ra₄₁ to         Ra₄₁₆ is selected from the group consisting of         —(CH₂)_(l)—SO₂—Z—SO₂—R, —(CH₂)_(l)—CO—Z—SO₂—R,         —(CH₂)_(l)—SO₂—Z—CO—R and —(CH₂)_(l)—N(R)—SO₃ ⁻,         where     -   l represents a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the substituents Ra₄₈ to Ra₄₁₁ form the remaining members of a carbo- or heterocyclic ring system which may contain up to 4 rings which may optionally carry a plurality of substituents. Ra₄₈ together with Ra₄₁₁ may form a π bond and Ra₄₉ and Ra₄₁₀ may represent substituents.

The following substituents may form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

The substituent Ra₄₅ together with Ra₄₆ or Ra₄₅ with Ra₄₄, or Ra₄₆ with Ra₄₁, and/or Ra₄₁ with Ra₄₂ and/or Ra₄₄ with Ra₄₃. Particularly preferred compounds of the formula 4a

in which

-   -   Ra₄₁ to Ra₄₁₁ independently of one another represent any         substituent,     -   Yb₄₁ and Yb₄₂ independently of one another substituted or         substituted C or N, where the substituents may also form a 5- or         6-membered aliphatic or aromatic carbo- or heterocycle and     -   n represents 0, 1, 2, 3, 4 or 5,     -   with the proviso that at least one of the substituents Rb₄₁ to         Rb₄₁₁ is selected from the group consisting of         —(CH₂)_(l)—SO₂—Z—SO₂—R, —(CH₂)_(l)—CO—Z—SO₂—R,         —(CH₂)_(l)—SO₂—Z—CO—R and —(CH₂)_(l)—N(R)—SO₃ ⁻,         where     -   l represents a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the following substituents form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

The substituent Rb₄₅ together with Rb₄₆ and/or Rb₄₅ with Rb₄₄, and/or Rb₄₆ with Rb₄₁, and/or Rb₄₁ with Rb₄₂ and/or Rb₄₄ with Rb₄₃.

The substituent Rb₄₉ together with Rb₄₁₀ and/or Rb₄₈, the substituent Rb₄₁₀ together with Rb₄₁₁ and/or the substituent Rb₄₈ together with Rb₄₇. Particularly preferred compounds of the formula 4b

in which

-   -   Rc₄₁ to Rc₄₁₀ independently of one another represent any         substituent,     -   Yc₄₁ and Yc₄₂ independently of one another substituted or         unsubstituted C or N, where the substituents may also form a 5-         or 6-membered aliphatic or aromatic carbo- or heterocycle,     -   Ac₄₁ represents O, S, Se, Te, N—Rc₄₁₁, C(Rc₄₁₂)(Rc₄₁₃) or         —C(Rc₄₁₄)═C(Rc₄₁₅)—, where Rc₄₁₁ to Rc₄₁₅ may represent H,         optionally substituted alkyl, aryl, or alkenyl, having up to 20         C atoms, or any substituent and     -   n may represent 0, 1, 2, 3, 4 or 5,     -   with the proviso that at least one of the substituents Rc₄₁ to         Rc₄₁₅ is selected from the group consisting of         —(CH₂)_(l)—SO₂—Z—SO₂—R, —(CH₂)_(l)—CO—Z—SO₂—R,         —(CH₂)_(l)—SO₂—Z—CO—R and —(CH₂)_(l)—N(R)—SO₃ ⁻,         where     -   l represents a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the following substituents may form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

The substituent Rc₄₅ together with Rc₄₆ or Rc₄₅ with Rc₄₄, or Rc₄₆ with Rc₄₁, and/or Rc₄₁ with Rc₄₂ and/or Rc₄₄ with Rc₄₃

The substituents Rc₄₇ to Rc₄₁₀ form the remaining members of a carbo- or heterocyclic ring system which may contain up to 4 rings which may optionally carry a plurality of substituents. Rc₄₇ together with Rc₄₁₀ may form a π bond and Rc₄₈ and Rc₄₉ may represent substituents. Compounds of the formula 4c which are to be used with particular preference

in which

-   -   Rd₄₁ to Rd₄₁₀ independently of one another represent any         substituent,     -   Yd₄₁ and Yd₄₂ independently of one another represent substituted         or unsubstituted C or N, where the substituents may also form a         5- or 6-membered aliphatic or aromatic carbo- or heterocycle,     -   n may represent 0, 1, 2, 3, 4 or 5     -   with the proviso that at least one of the substituents Rd₄₁ to         Rd₄₁₀ is selected from the group consisting of         —(CH₂)_(l)—SO₂—Z—SO₂—R, —(CH₂)_(l)—CO—Z—SO₂—R,         —(CH₂)_(l)—SO₂—Z—CO—R and —(CH₂)_(l)—N(R)—SO₃ ⁻,         where     -   l represents a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the following substituents may form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

The substituent Rd₄₅ together with Rd₄₆ and/or Rd₄₅ with Rd₄₄, and/or Rd₄₆ with Rd₄₁, and/or Rd₄₁ with Rd₄₂ and/or Rd₄₄ with Rd₄₃.

The substituent Rd₄₉ together with Rd₄₁₀, and/or the substituent Rd₄₈ together with Rd₄₇. Compounds of the formula 4d which are to be used with particular preference

5. Squaric Acids

For the purpose of the present invention, squaric acids that are to be used with preference are those of the formula 5a, 5b and 5c

in which

-   -   Ra₅₁ to Ra₅₁₀ independently of one another may represent any         substituent,     -   Ya₅₁, Ya₅₄ independently of one another substituted or         unsubstituted C or N, where the substituents may also form a 5-         or 6-membered aliphatic or aromatic carbo- or heterocycle.     -   s and t independently of one another represent 0, 1, 2 or 3,     -   k represents 1 or 2,     -   Ya₅₂ represents O, S, or ═NRa₅₁₁,     -   Ya₅₃ represents O—Ra₅₁₂, S—Ra₅₁₃ or N(Ra₅₁₄)(Ra₅₁₅),     -   where Ra₅₁₁ to Ra₅₁₅ independently of one another may represent         any substituent     -   Aa₅₁ and Ba₅₁ independently of one another represent O, S, Se,         Te, N—Ra₅₁₆, C(Ra₅₁₇)(Ra₅₁₈) or —C(Ra₅₁₉)═C(Ra₅₂₀)—, where Ra₅₁₆         to Ra₅₂₀ may represent any substituent, preferably H, optionally         substituted alkyl, aryl or alkenyl, having up to 20 C atoms,     -   with the proviso that at least one of the substituents Ra₅₁ to         Ra₅₂₀ selected from the group consisting of —(CH₂)₁—SO₂—Z—R,         —(CH₂)₁—CO—Z—SO₂—R, —(CH₂)₁—SO₂—Z—CO—R and —(CH₂)₁—N(R)—SO₃ ⁻,         where     -   1 is a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the substituents Ra₅₂ to Ra₅₅ or Ra₅₆ form the remaining members of a carbo- or heterocyclic ring system which may contain up to 4 rings which may optionally carry a plurality of substituents. Ra₅₂ together with Ra₅₅ and/or Ra₅₆ together with Ra₅₉ may form a π bond and Ra₅₃ and Ra₅₄ or Ra₅₇ and Ra₅₈ may represent substituents. Particularly preferred compounds of the formula 5a

in which

-   -   Rb₅₁ to Rb₅₁₂ independently of one another may represent any         substituent,     -   Yb₅₁ to Yb₅₄ independently of one another substituted or         unsubstituted C or N, where the substituents may also form a 5-         or 6-membered aliphatic or aromatic carbo- or heterocycle.     -   s and t independently of one another represent 0, 1, 2 or 3,     -   k represents 1 or 2,     -   Yb₅₂ represents O, S, or ═NRb₅₁₃,     -   Yb₅₃ represents O—Rb₅₁₄, S—Rb₅₁₅ or N(Rb₅₁₆)(Rb₅₁₇),     -   where Rb₅₁₃ to Rb₅₁₇ independently of one another may represent         any substituent,     -   with the proviso that at least one of the substituents Rb₅₁ to         Rb₅₁₇ is selected from the group consisting of         —(CH₂)₁—SO₂—Z—SO₂—R, —(CH₂)₁—CO—Z—SO₂—R, —(CH₂)₁—SO₂—Z—CO—R and         —(CH₂)₁—N(R)—SO₃ ⁻,         where     -   1 is a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the following substituents may form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

-   -   The substituent Rb₅₅ together with Rb₅₆ and/or Rb₅₅ and Rb₅₄,         and/or Rb₅₆ with Rb₅₁, and/or Rb₅₁ with Rb₅₂ and/or Rb₅₄ with         Rb₅₃.

The substituent Rb₅₉ together with Rb₅₈ or Rb₅₁₀ together with Rb511, the substituent Rb₅₁₁ together with Rb₅₁₂ and/or the substituent Rb₅₈ together with Rb₅₇. Particularly preferred compounds of the formula 5b

in which

-   -   Rc₅₁ to Rc₅₁₁ independently of one another may represent any         substituent     -   Yc₅₁ to Yc₅₄ independently of one another substituted or         unsubstituted C or N, where the substituents may also form a 5-         or 6-membered aliphatic or aromatic carbo- or heterocycle.     -   s and t independently of one another represent 0, 1, 2 or 3,     -   k represents 1 or 2,     -   Yc₅₂ represents O, S or ═NRc₅₁₂,     -   Yc₅₃ represents O—Rc₅₁₃, S—Rc₅₁₄ or N(Rc₅₁₅)(Rc₅₁₆),     -   Ac₅₁ represents O, S, Se, Te, N—Rc₅₁₇, C(Rc₅₁₈)(Rc₅₁₉) or         —C(Rc₅₂₀)═C(Rc₅₂₁)—, where Rc₅₁₂ to Rc₅₂₁ represent any         substituent, preferably H, optionally substituted alkyl, aryl or         alkenyl, having up to 20 C atoms,     -   with the proviso that at least one of the substituents Rc₅₁ to         Rc₅₂₁ is selected from the group consisting of         —(CH₂)₁—SO₂—Z—SO₂—R, —(CH₂)₁—CO—Z—SO₂—R, —(CH₂)₁—SO₂—Z—CO—R and         —(CH₂)₁—N(R)—SO₃ ⁻,         where     -   1 is a number between 1 and 6,     -   Z represents NH or N⁻ and     -   R preferably represents optionally substituted alkyl or aryl.

In a preferred embodiment, the following substituents may form a 5- or 6-membered carbo- or heterocycle which is optionally also fused:

The substituent Rc₅₅ together with Rc₅₆ and/or Rc₅₅ with Rc₅₄, and/or Rc₅₆ with Rc₅₁, and/or Rc₅₁ with Rc₅₂ and/or Rc₅₄ with Rc₅₃.

The substituents Rc₅₇ to Rc₅₁₀ preferably from the remaining members of a carbo- or heterocyclic ring system which may contain up to 4 rings which may optionally carry a plurality of substituents. Rc₅₈ together with Rc₅₉ may form a π bond and Rc₅₇ and Rc₅₁₀ may represent substituents. Particularly preferred compounds of the formula 5c

For the purpose of the present application, substituents are to be understood as meaning, for example, halogens, such as, in particular F, Cl or Br, furthermore alkoxy, alkylthio, aryloxy, arylthio, acylamino, alkylsulfonamido, alkylsulfamoyl, alkylcaramoyl, arylsulfonamido, arylsulfamoyl, arylcarbamoyl, alkyl, alkenyl, aryl, hetaryl, arylene, hetarylene, alkylene, alkoxycarbonyl, ureido or cyano groups. Preference is furthermore given to substituted and also non-substituted cycloalkyl and also to substituted and non-substituted aryl, in particular phenyl groups; particularly preferred substituents are optionally substituted alkyl, aryl, sulfoalkyl, carboxyalkyl, —(CH₂)₁—SO₂—Z—SO₂—R, —(CH₂)₁—CO—Z—SO₂—R, —(CH₂)₁—SO₂—Z—CO—R, —(CH₂)₁—N(R)—SO₃ ⁻, where 1 represents a number between 1 and 6 and Z represents NH or N⁻ and R preferably represents optionally substituted alkyl or aryl.

For the purpose of the present invention, alkenyl is to be understood as meaning linear or branched, cyclic or straight-chain, substituted or non-substituted unsaturated hydrocarbon radicals, such as, for example, ethenyl, 2-propenyl, isopropenyl.

For the purpose of the present application, carbo- or heterocyclic ring systems are to be understood as meaning ring systems consisting of, preferably 4, 5, 6, 7 or 8 carbon atoms, where up to 3 carbon atoms may be replaced by heteroatoms such as, in particular, N, O, SE or S. The ring systems may be aliphatic or aromatic rings; preferably, 2, 3 or 4 identical or different rings may also be present as a fused system. For the purpose of the present application, carbocyclic aromatic groups are to be understood as meaning, preferably, 5- or 6-membered fused and/or substituted ring systems, in particular phenyl and naphthyl. Unsaturated heterocyclic groups are to be understood as meaning, preferably, 5- or 6-membered ring systems which may be present in monocyclic form, but also as fused ring systems. Here, suitable heteroatoms are, in particular, N, S and O. A ring system may preferably have between 1 and 3 heteroatoms, the heteroatoms being identical or different. Preference is given to: furyl, indolyl, thienyl, pyrrolyl and carbazolyl. These groups may be unsubstited or, preferably, substituted. Here, it is also possible for carbocyclic aromatic groups and unsaturated heterocyclic groups for their part to be present as substituents, which is also meant to include the possible fusion of different ring systems.

Compounds of one of the formulae 1 to 5 which are particularly preferred according to the invention are listed below:

According to the invention, the dyes can also be conjugated to antibodies and used, for example, in flow cytometry. One advantage of these polymethine dyes is the fact that a dye can be tailored for virtually any wavelength between 500 and 900 nm. As a result, multicolor detection is also possible. In flow cytometry, for example, different antibodies can in each case be labeled with one fluorescent dye and then be detected simultaneously in an experiment using a suitable detection system. The specific antibodies then identify different epitopes on the cells. Using this system, it is also possible to quantify the analytes. Thus, the use of a plurality of fluorescently labeled antibodies gives a higher information content per experiment. This means a cost advantage, in particular, for example, for clinical tests.

Furthermore, it is also possible to carry out qualitative and/or quantitative detection methods by labeling of biomolecules which are interacting with each other by fluorescence resonance energy transfer.

The present invention also provides the use of the dyes for labeling DNA, RNA or nucleotide analogs such as PNA, and to this end, they can be used in nucleic acid assays. Here, usually, activated dye molecules are bound to nucleotides, nucleosides, nucleotide analogs or oligonucleotides or analogs thereof. Binding takes place via a nucleophilic group of the nucleotides or analogs which reacts with the activated dye. Customary nucleophilic groups are amino groups, thiol groups, hydoxyl groups or other groups. The activated groups are generally derivatives of carboxyl groups, such as N-hydroxysuccinimidates, isothiocyanates, maleimides or iodoacetamide derivatives. Moreover, a nucleophilic groups may react with a phosphoamidite radical, resulting in a covalent bond between the dye molecule and the biomolecule.

In further applications, the dyes can also be attached to solid phases, such as, for example, polymeric beads, or be incorporated into the same. The polymeric beads can be functionalized and for their part act as fluorescent labels in bioanalysis.

As already described, polymethine dyes which contain a group of the formula (I) are already known from photography; they are obtainable, for example, as described in EP 0 534 283, EP 0 530 511, DE 1 081 311.

In contrast, polymethine dyes which contain a group of the formula (I) and at the same time a group which renders them capable of forming covalent bonds, are selected from the group of the following compounds:

and are novel and have not yet been described in the prior art. Accordingly, hereinbelow synthesis procedures for obtaining such “activated” dyes are described:

Synthesis procedures for preparing the dyes F and J according to the invention. 1. Synthesis of F

A

For 12 h, 0.15 mol of 3,4-dihydroxy-3-cyclobutene-1,2-dione in 150 mL of absolute ethanol is heated under reflux.

The reaction mixture is concentrated.

The oil is purified by column chromatography.

Mobile phase: methyl tert-butyl ether/iso-octane 50:50

Stationary phase: AMICON 35-70 mic.

Yield: 48%

C

0.055 mol of A, 0.0068 mol of B and 0.0078 mol of triethylamine in 50 mL of absolute ethanol are boiled at reflux. After 35 min, 0.107 mol of triethylamine is added. After 3.5 h, the mixture is cooled. Ethyl acetate is added and, after vigorous stirring, the mixture is decanted. These process steps are repeated a further 3 times. This gives an oil (C).

Hydrolysis to D

The oil C in 0.145 mol of (C₄H₉)₄NOH (1 M in methanol) is heated under reflux. After 30 min, the mixture is cooled. 500 ml of acetone and 16 mL of concentrated HCl are added. The precipitated solid is filtered off. Yield: 28 g

E

0.63 mol of 2,3,3-trimethylindolenine and 0.5 mol of Br(CH₂)₅COOH in a small amount of acetonitrile are heated under reflux for 3 h. The temperature of the reaction mixture is 130° C. 0.5 mol of acetic acid are added and the mixture is cooled to 100° C. 250 mL of methyl ethyl ketone are then added, and the mixture is further cooled in an ice bath to 15° C. The precipitated solid is filtered off and dried at 50° C. Yield: 75%

G

3.5 mol of phenylhydrazine-4-sulfonic acid and 1.9 L of acetic acid are heated at 105° C. Over the course of 30 min, 4.8 mol of 3-methyl-2-butanone are added dropwise. After 1 h 50 min, heating is removed and 4.2 mol of potassium acetate in 3.5 L of methanol are added. The mixture is further cooled in an ice bath and the precipitated solid is filtered off. It is washed with ethyl acetate. Yield: 77%

H

26.8 mol of chloroacetyl chloride and 25.5 mol of methanesulfonamide in 10.2 L of butyl acetate are slowly boiled at reflux. After 8 h, the mixture is cooled to 20° C. The precipitated solid is filtered off and stirred in 4 L of butyl acetate. The precipitated solid is dried at 50° C. in a fan-assigned drying cabinet. Yield 74%

B

2.5 mol of G and 2.5 mol of H in 1.3 L of sulfolane are heated at 130° C. After 5 h and 50 min, the mixture is cooled to 50° C. 2.5 L of acetone are added and the mixture is further cooled to room temperature. The precipitated solid is filtered off and washed with acetone. The precipitated solid is stirred in 1.6 L of warm demineralized water. 1.6 L of ethanol are added and the mixture is cooled. The precipitated solid is filtered off and washed with ethanol. Yield: 53%

F

In a flask fitted with a Dean-Stark separator, 4.7 g of D, 3.5 g of E and 1.4 mL of triethylamine in n-butanol/toluene are boiled at reflux. After 2.5 h, the mixture is cooled. The mixture is extracted 3 times with water. The reaction mixture is concentrated and then redissolved in ethanol. 3 g of potassium acetate are added and the mixture is filtered off. The precipitated solid is taken up in water, and acetone is added. The solvent is decanted off and more acetone is added. These process steps are repeated until precipitation occurs. The precipitated solid is filtered off. 2. Synthesis of J

I

0.1 mol of G and 0.12 mol of Br(CH₂)₅COOH in 35 mL of sulfolane are heated at 130° C. After 3 h, 100 mL of dimethylacetamide are added and the mixture is cooled to room temperature. The precipitated solid is filtered off and, on the filter, washed with dimethylacetamide. The precipitated solid is boiled in acetone. After cooling, the mixture is filtered and the product is dried at 50° C. in a fan-assisted drying cabinet. Yield: 90%

J

For 2 h and 10 min, 4.7 g of D and 3.5 g of I in 80 mL of n-butanol/pyridine (7:1) are boiled at reflux. The mixture is cooled to room temperature and the precipitated solid is filtered off. On the filter, the solid is washed with n-butanol or acetone. The precipitated solid is taken up in 7 mL of demineralized water and 30 ml of acetonitrile are added with stirring. The liquid phase is decanted off and, with stirring, more acetonitrile is added. The liquid phase is decanted off and the oil is stirred with 40 mL of a saturated sodium chloride solution. The fine suspension is centrifuged and the liquid phase is decanted off. The precipitated solid is dissolved in methanol and the sodium chloride is filtered off. The filtrate is concentrated. The product is dried at 50° C. Yield: 1.57 g (18.5%)

General Procedure for Preparing Succinimidyl Esters from Polymethine Dyes Having Carboyl Functions

The present procedure describes the preparation of succinimidyl esters from polymethine dyes having at least one carboxyl function. These esters can then be used for labeling biomolecules such as proteins, antibodies or nucleic acids.

0.05 mmol of dye and disuccinimidyl carbonate (1.5 equivalents per carboxyl group) are dissolved in 40 mL of DMF, and a spatula tip of dimethylaminopyridine is added. The mixture is stirred at 40° C. for 3 hours. After cooling, the supernatant is, in 1 ml aliquots, divided into glass vials. The solvent is evaporated under reduced pressure and the product is stored until use at 4° C. in the dark.

The crude product is used directly for labeling proteins.

General Procedure for Labeling Proteins

For labeling, the crude product (1 aliquot) is dissolved in 1 ml of DMF. 50 μl of this solution are pipetted to 1.5 mL (=1 mg) of an anti-HSA solution in 0.1 M carbonate buffer pH 9.2. The reaction solution is stirred at RT for 2 hours.

Using gel permeation chromatography over a Sephadex G50 column which had earlier been equilibrated with PBS, pH 7.4, the conjugate is then separated from unbound dye.

General Procedure for Labeling Oligonucleotides with Dye

The dyes can also be used for labeling DNA or RNA nucleotides or nucleotide analogs.

50 mmol of activated dye, for example isothiocyanate K, are dissolved in 50 ml of DMF or acetonitrile. 10 nmol of NH2-oligonucleotide are initially charged in 150 μl of 50 mM carbonate buffer, pH 9.2, and 75 nmol of dye are slowly added using a pipette. The solution was stirred at 25° C. under the exclusion of light for 3 to 5 hours. Purification of the Oligonucleotide

Purification of the labeled oligonucleotide from unbound dye and unlabeled DNA was carried out by RP-HPLC over a C8 column. Elution was carried out using a linear gradient, for example from 70% water to 100% methanol over 30 min.

The fractions which absorbed simultaneously at 254 nm and the absorption maximum of the dye were collected, and the solvent was removed under reduced pressure.

For further use of the modified oligonucleotide, this may be separated from excess salts by ethanol precipitation.

EXAMPLES

Table 1 shows the relative quantum yields of the dyes at similar molar dye/protein ratios. The protein used was HSA (Human Serum Albumin).

TABLE 1 Extinction coefficient Dye D/P ratio rel. Q.Y. [mol-1*cm²* Sq-1 (Ex.) 0.36 0.64 172 000 Sq-2 (Ex.) 0.40 1.0 234 000 F (inv) 0.37 1.35 420 000 J (Inv) 0.46 1.36 240 000 Determination of the Molar Dye/protein Coefficient

The protein concentration is determined using the BCA method of Pierce (literature: Bradford, M. M.; Anal. Biochem. (1976) 72, 248-254). A calibration curve for anti-HSA had been prepared beforehand.

The dye concentration is determined via the absorption spectrum.

Determination of the Quantum Yield

The quantum yield was determined relative to Cy5

Determination of the Extinction Coefficient

The extinction coefficient was determined assuming that Beer's law applies.

Labeling of Activated Dye F to Anti-HAS

For labeling, the crude product (1 aliquot) is dissolved in 1 ml of DMF. 50 μl of this solution are pipetted to 1.5 mL (=1 mg) of anti-HSA solution in carbonate buffer pH 9.2. The reaction solution is stirred at RT under the exclusion of light for 2 hours.

Using gel permeation chromatography over a Sephadex G25 column, the conjugate is then separated from unbound dye. The elution buffer used is PBS, pH 7.4.

Protein conjugates of other dyes, which are listed in Table 1, were prepared in an analogous manner.

Table 1 shows that the dyes according to the invention have higher quantum yields and extinction coefficients than, for example, Cy5. Accordingly, they are particularly suitable for use as fluorescent markers in bioanalysis.

The dye conjugates of the dyes according to the invention show a lower tendency to form aggregates than polymethine dyes which have hitherto been customary. This becomes evident in Table 2.

TABLE 2 Absorption monomer/absorption dimer Water 2.0 M NaCl 3.8 M NaCl CY5 3.12 3.18 3.19 Sq-3 2.98 2.14 1.78 Sq-1 4.65 4.48 3.40 Sq-2 2.96 2.98 1.36 F 2.89 2.81 2.81 J 4.40 4.35 4.40

The intensity of the absorption in the absorption maximum of the monomer is a relative measure for the concentration of the monomer in the solution, the same also applies to the concentration of the aggregate, this can be correlated to the intensity of the absorption in the absorption maximum of the aggregate, which is shifted hypsochronically compared to the monomer.

Table 2 shows the ratio of the intensities of the absorption maxima of the monomer and aggregates. The increasing salt concentration causes an aggregate formation. Compared to CY5, the dye J according to the invention shows a considerably lower aggregate formation. Squaric acid dyes such as Sq-1 to Sq-3 have no stable characteristics with respect to aggregate formation, which increases with increasing salt concentration.

In contrast, the dyes F and J according to the invention have stable characteristics.

Photostability

The photostability was determined by exposing dye solutions to daylight. The absorption of the solutions varied between 1.4 and 1.6 O.D. The reduction of the absorption over time is a measure for the photostability of the dyes. These conditions were chosen since the handling of the dyes, too, includes manual process steps during which the dyes are exposed to daylight. In the case of CY5, it is clearly noticeable that, even after 30 min, the absorption has decreased by 12%. This is not the case for the dyes according to the invention, and the latter are thus also more user-friendly, by maintaining a stable absorption. 

1. A polymethine dye, comprising at least one substituent of the formula (I)

in which n represents 1, 2, 3, 4, 5, 6, 7, 8 or 9 Y, A represent C═O or —SO₂, and R represents an optionally substituted alkyl or aryl radical, and at least one substituent selected from the group consisting of


2. A polymethine dye as claimed in claim 1, wherein the polymethine dye is at least one substance selected from the group consisting of a cyanine dye, merocyanine, rhodacyanine, a styrene dye, a squaric acid dye and a crotonic acid dye.
 3. A method of using the polymethine dye as claimed in claim 1 wherein said dye is contacted with at least one biomolecule to label the biomolecule by covalent binding.
 4. A method of using the polymethine dye as claimed in claim 1, wherein the dye is contacted with at least one biomolecule to stain at least a portion of the biomolecule.
 5. A method of using at least one of the following compounds

wherein said compounds are contacted with at least one biomolecule to label at least a portion of the biomolecule. 